: The long-term goal of this project is to elucidate structural and functional features of nicotinic acetylcholine receptors containing a7, or8 or or9 subunits (a7-, a8-, or a9-nAChR). a7, a8 and a9 are the most ancient nAChR subunits, and the homomeric complexes that they are postulated to create are perhaps the most simple form of nAChR. Nevertheless, these nAChR, particularly a7-nAChR, are widespread mediators of classical excitatory neurotransmission. They also may play novel physiological roles as modulators of neurotransmitter release, neurite outgrowth, and even neuronal death/survival. a7-nAChR also have been implicated in development, differentiation, and disease of the nervous system and are targets of tobacco nicotine action. The project is founded in preliminary work demonstrating expression of these subunits as homomeric nAChR in the native nAChR-null human epithelial cell line SH-EP1 and revealing startling properties of wild-type and mutant forms of these nAChR. The specific aims of the project are (1) to generate and characterize mutant forms of a7-nAChR that recognize nicotine as a functional antagonist, (2) to generate and characterize functional forms of truncated a7nAChR, (3) to ascertain whether nAChR containing oc7 subunits can also assemble as heteromers and whether such heteromeric a7-nAChR have distinctive properties, and (4) to characterize heterologously expressed forms of a8nAChR and a9-nACh R. These studies will test hypotheses that selected mutation(s) of ligand binding and/or channel lining domains of nAChR influence whether drugs act as agonists or antagonists. They will test the hypothesis that truncated forms of nAChR subunits can be engineered to nevertheless retain abilities to assemble as ligand-binding, functional ion channels. The project will also test the hypothesis that a7 subunits only assemble as homomers. It will elucidate nAChR function when expressed in the same cellular environment and whether/how ceil environment influences expression of nAChR. The planned studies will help to reveal structural features critical to ligand recognition, assembly, and function of nACh R. Findings in the project are relevant to our understanding of the important roles played by nAChR in nervous system function and disease and in nicotine dependence.